Multiplex immunoassays for hemoglobin, hemoglobin variants, and glycated forms

ABSTRACT

Hemoglobin, its variants, and glycated forms of each are determined individually in a multiplex assay that permits correction of the measured level of HbA1c to account for glycated variants and other factors related to the inclusion of the variants in the sample. New antibodies that are particularly well adapted to the multiplex assay are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.15/441,455, filed Feb. 24, 2017, which is a continuation of U.S.application Ser. No. 14/947,939, filed Nov. 20, 2015, issued as U.S.Pat. No. 9,605,060, which is a divisional application of U.S.application Ser. No. 14/081,799, filed Nov. 15, 2013, issued as U.S.Pat. No. 9,213,034, which is a continuation application of U.S.application Ser. No. 12/941,738, filed Nov. 8, 2010, issued as U.S. Pat.No. 8,603,828, which claims the benefit of U.S. Provisional PatentApplication No. 61/262,488, filed Nov. 18, 2009, each of whichapplications is herein incorporated by reference.

REFERENCE TO A “SEQUENCE LISTING” SUBMITTED AS ASCII TEXT FILES VIAEFS-WEB

The Sequence Listing written in file095191-087450US-1106887_SequenceListing.txt created on Sep. 27, 2018,16,092 bytes, machine format IBM-PC, MS-Windows operating system, inaccordance with 37 C.F.R. §§ 1.821- to 1.825, is hereby incorporated byreference in its entirety for all purposes.

BACKGROUND OF THE INVENTION 1. Field of the Invention

This invention lies in the field of assays for glycated hemoglobin.

2. Description of the Prior Art

For individuals suffering from type 1 or type 2 diabetes mellitus,maintenance of glycemic control is of prime importance, and suchmaintenance requires the determination of the level of hemoglobin A_(1c)in the blood of these individuals. With diabetes reaching globalepidemic proportions, it is particularly important to have accurate andreproducible HbA_(1c) assays. HbA_(1c) assays are also used in thescreening of individuals for diabetes.

HbA_(1c) measurements for both patient monitoring and screening aretaken as an average over the lifetime of an erythrocyte. This average iscompromised by several physiological conditions, notable among which arethe presence of hemoglobin variants and thalassemias in the patient'sblood. Hemoglobin variants are prevalent among certain ethnic groups andin certain geographical regions. Of the over 800 variants knownworldwide, the most common are HbS, HbC, HbD, and HbE. HbS is mostprevalent among individuals of African descent, HbD among individuals ofPunjabi Indian descent, and HbE among individuals of Southeast Asia.Other known forms of hemoglobin are HbF (fetal hemoglobin) and HbA2,both of which can be elevated in thalassemia, a relatively commoncondition characterized by an imbalance of hemoglobin alpha and betasubunits. Beta thalassemias can also occur in the presence of HbE andHbS, and the combined sickle/beta thalassemia trait occurs mostfrequently among individuals of Mediterranean descent. Variants andthalassemias can cause inaccuracies in HbA_(1c) measurements byaffecting such factors as red blood cell survival and glycosylationrates. Variants also affect immunologically determined levels ofglycated hemoglobin since immunoreactivity differs from one glycatedvariant to the next and also between glycated variants and HbA itself.Health care professionals must therefore know of the presence ofvariants and their proportions relative to HbA as well as the presenceof thalassemias to achieve a proper determination of glycemic control.

Determinations of hemoglobin variants are typically done separately fromdeterminations of HbA_(1c) regardless of whether a variant is actuallyknown to be present.

Antibodies to specific variants have been developed for this purpose,and the following is a sampling of reports on such antibodies:

-   HbS: Jensen, R. H., et al., “Monoclonal antibodies specific for    sickle cell hemoglobin,” Hemoglobin 9(4), 349-362 (1985)-   HbS: Epstein, N., et al., “Monoclonal antibody-based methods for    quantitation of hemoglobins: application to evaluating patients with    sickle cell anemia treated with hydroxyurea,” Eur. J. Haemotol.    57(1), 17-24 (1996)-   HbA: Rosenthal, M. A., et al., “Binding specificity of a monoclonal    antibody to human HbA,” Hemoglobin 19(3-4), 191-196 (1995)-   HbS and HbC: Garver, E. A., et al., “Screening for hemoglobins S and    C in newborn and adult blood with a monoclonal antibody in an ELISA    procedure,” Annals of Hematology 60(6), 334-338 (1990)-   Hb with single amino acid substitutions: Stanker, L. H., et al.,    “Monoclonal antibodies recognizing single amino acid substitutions    in hemoglobin,” J. Immunol. 136 (11), 4174-4180 (1986)-   Hb variants: Moscoso. H., et al., “Enzyme immunoassay for the    identification of hemoglobin variants,” Hemoglobin 14(4), 389-98    (1990)-   Hb variants: Schultz, J. C., “Utilization of monoclonal    antibody-based assay HemoCard in screening for and differentiating    between genotypes of sickle cell disease and other    hemoglobinopathies,” J. Clin. Lab. Anal. 9(6), 366-374 (1995)

Despite these reports and others, determinations of variants arepresently performed by either high performance liquid chromatography(HPLC) or electrophoresis. HPLC can indeed be a rapid means of obtainingthe HbA_(1c) level, but extended HPLC gradients are needed for detectingand quantifying the variants and thalassemias, since in HPLC impuritiesco-elute with the variants, and different variants tend to co-elute witheach other. In fact, certain variants cannot be resolved by HPLC, evenwith the most optimized HPLC gradients. Typically, separate HPLC methodsfor rapid A_(1c) measurements and variant and thalassemia testing areused, therefore making it impossible to simultaneously determine theA_(1c) level and variant or thalassemia status by HPLC, much less in arapid manner.

Assays that provide simultaneous detection of multiple analytes aretermed “multiplex” assays, and disclosures of multiplex assays usingaffinity-type binding reactions on the surfaces of beads that are thendetected by flow cytometry are disclosed in the following patents:

-   Watkins, M. I., et al., “Magnetic particles as solid phase for    multiplex flow assays,” U.S. Pat. No. 6,280,618 B2, issued Aug. 28,    2001-   Watkins, M. I., et al., “Magnetic particles as solid phase for    multiplex flow assays,” U.S. Pat. No. 6,872,578 B2, issued Mar. 29,    2005-   Thomas, N., “Multiple assay method,” U.S. Pat. No. 6,913,935 B1,    issued Jul. 5, 2005-   Hechinger, M., “Platelet immunoglobulin bead suspension and flow    cytometry,” U.S. Pat. No. 6,933,106 B1, issued Aug. 23, 2005-   Hechinger, M., “Anti-platelet immunoglobulin bead positive control,”    U.S. Pat. No. 6,951,716 B1, issued Oct. 4, 2005-   Watkins, M. I., et al., “Multi-analyte diagnostic test for thyroid    disorders,” U.S. Pat. No. 7,271,009 B1, issued Sep. 8, 2007-   Bell, M. L., “Assay procedures and apparatus,” U.S. Pat. No.    7,326,573 B2, issued Feb. 5, 2008-   Song, Y., et al., “Multiplex protein interaction determinations    using glutathione-GST binding,” US 2002/0115116 A1, published Aug.    22, 2002

The success of multiplex assays for certain combinations of analytesdoes not however provide assurance, or even a high level of expectation,that similar success will be achieved for all combinations of analytes,particularly combinations with a high level of homology among theanalytes. Hemoglobin and its variants and glycated forms are one suchcombination. Multiplex assays involve a plurality of differentimmunoreactants in intimate mixture in a common reaction medium, whichcreates competition among the immunoreactants for the differentanalytes, more so than in media where a single immunoreactant ispresent, and the cross-reactivities occur in multiple directions. Thebead sets themselves must also be differentiated at the same time as theimmunoassays are performed. This differentiation, whether by the use ofdifferent dyes on different bead sets, a different size for each beadset, or other known differentiation factors, adds a further level ofcomplexity and further opportunities for cross-talk.

SUMMARY OF THE INVENTION

The present invention resides in the discovery that hemoglobin variantscan be differentiated from each other and from HbA_(1c) and from totalhemoglobin, and the levels of each measured, in a multiplex immunoassay.The assay can, e.g., detect a single variant in addition to HbA_(1c) andtotal hemoglobin or two or more variants and total hemoglobin. When twoor more variants are detected, different combinations of variants can beselected, although preferably the assay will include the four mostcommon variants, HbS, HbC, HbE, and HbD. The assay may also include themeasurement of HbA2 and HbF. The invention thus resides in a method fordetecting and identifying the presence of hemoglobin variants in apatient's blood. The invention also resides in a method for measuringthe level of HbA1c relative to total hemoglobin while correcting theresult for the presence of variants that may also be present. Here aswell, the correction can be for individual variants or combinations ofvariants. The invention also resides in a method for the simultaneousdetection of A_(1c) and hemoglobin variants without correction, which isuseful in certain cases. A still further aspect of the invention is themeasurement of levels of particular variants in glycated form. When avariant is known to be present, the glycated version of that variant canbe measured and added to the level of HbA_(1c) to obtain an accurateindication of total glycated hemoglobin.

In a further aspect, the invention provides antibodies having selectivebinding affinity for hemoglobin variants that can be used in the methodsof the invention. In some embodiments, the invention provides amonoclonal antibody that has selective binding affinity for HbC andglycated HbC, wherein the monoclonal antibody binds to an HbC minimalepitope ⁴TPKEKSAVT¹² (SEQ ID NO:1); or to an HbC minimal epitopecomprising the amino acid sequence TX₁KE or LTX₁KE (SEQ ID NO:2),wherein X₁ is one of the 20 common naturally occurring amino acids. Insome embodiments, the invention provides a monoclonal antibody havingselective binding affinity for HbS and glycated HbS, wherein themonoclonal antibody binds to an HbS minimal epitope ³LTPVEKSAVT¹² (SEQID NO:3); or to an HbS minimal epitope comprising the amino acidsequence PVEX₂X₃A (SEQ ID NO:4) or LTPVEX₂X₃A (SEQ ID NO:5), whereineach of X₂ and X₃ is an amino acid independently selected from the 20common naturally occurring amino acids. In some embodiments, theinvention provides a monoclonal antibody having selective bindingaffinity for HbE and glycated HbE, wherein the antibody binds to an HbEminimal epitope ²²EVGGK²⁶ (SEQ ID NO:6); or to an HbE minimal epitopecomprising the amino acid sequence DEVGGK (SEQ ID NO:7) or EVX₄X₅K,wherein each of X₄ and X₅ is an amino acid independently selected fromthe 20 common naturally occurring amino acids. In some embodiments, theinvention provides a monoclonal antibody having selective bindingaffinity for HbD and glycated HbD, where the monoclonal antibody bindsto an HbD minimal epitope ¹²¹QFTPP¹²⁵ (SEQ ID NO:8); or to an HbDminimal epitope comprising the amino acid sequence GX₆QFX₇PP (SEQ IDNO:9) or QFX₇PP (SEQ ID NO:10), wherein each of X₆ and X₇ is an aminoacid independently selected from the 20 common naturally occurring aminoacids.

The invention also may employ an antibody, either a polyclonal antibodyor monoclonal antibody that selectively binds total hemoglobin (incomparison to non-hemoglobin polypeptides). In some embodiments, apan-reactive polyclonal antibody for use in the invention binds to oneor more epitopes present in the following regions of alpha globin andbeta globin: alpha globin ⁴⁹SHGSAQVKGHGKKVADALTNAVAHVDDMPNALSALSDHLHAHKLRRVDPV⁹⁶ (SEQ ID NO:11); beta globin ¹⁵WGKVNVDEVGGEALG³⁰ (SEQ IDNO:12), ⁴⁵FGDLSTP⁵¹ (SEQ ID NO:13), and ⁷⁶AHLDNLKGTFAT⁸⁷ (SEQ ID NO:14).In some embodiments, a pan reactive antibody is a monoclonal orpolyclonal antibody that binds to the epitope ⁹SAVTALWGKVNV²⁰ (SEQ IDNO:15) (beta globin) or ⁸KSAVTALWGKVNV²⁰ (SEQ ID NO:16) or ¹¹VTALW¹⁵(SEQ ID NO:17) or to a beta globin minimal epitope that comprises thesequence ALWG (SEQ ID NO:18) or VTX₉LW (SEQ ID NO:19), wherein X₉ is oneof the 20 common naturally occurring amino acids. An antibody for use inthe invention may bind to an epitope on beta globin, e.g.⁸KSAVTALWGKVNV²⁰ (SEQ ID NO:16), ⁵⁸PKVKAHGKKVLGAF⁷¹ (SEQ ID NO:20) or⁸⁷TLSELHCDKLHVDPENFR¹⁰⁴ (SEQ ID NO:21) (beta globin).

The invention further provides monoclonal antibodies that selectivelybind to glycated forms of hemoglobin, including binding to both normaland variant hemoglobins, but do not bind to non-glycated forms ofhemoglobin. A glycosylated residue ¹V and residue ²H are typicallyimportant for binding for such antibodies.

The method of the invention may additionally comprise detecting otherhemoglobin variants using antibodies, e.g., monoclonal antibodies, thatselectively bind such variants.

In typical embodiments, an antibody for use in the invention has a K_(D)that is anywhere in the range of from about 10⁻⁶ M to about 10⁻¹² M. Insome embodiments, the antibody has a K_(D) that is anywhere in the rangeof from about 10⁻⁷M to about 10⁻¹¹ M. In other embodiments, the antibodyhas a K_(D) anywhere in the range of about 10⁻⁸M to about 10⁻¹⁰ M.Typically the K_(D) is in the nM range, e.g., anywhere from about 10⁻⁹Mto about 10⁻¹⁰ M.

These and other features, objects, and advantages of the invention willbe better understood from the description that follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a schematic depicting an example of a sandwichimmunoassay for measuring glycated hemoglobin and hemoglobin variants.

FIG. 2 is a plot of comparative data between a series of multiplexassays in accordance with the present invention and a series of HPLCassays.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

The hemoglobin variants to be detected by the present invention are anyof the known variants reported in the literature or otherwise known toclinicians and researchers skilled in technology of hemoglobin, glycatedhemoglobin, and diabetes. As noted above, the four most commonhemoglobin variants are HbS, HbC, HbE, and HbD, although other variantscan be detected in addition to these four or in place of one or more ofthem. For example, two variants that are elevated in beta thalassemiaare HbF and HbA₂. The binding members used for each of these variants inthe multiplex assay are generally monoclonal antibodies, preferablythose that are developed expressly for the multiplex assay. Theantibodies preferably bind to epitopes on the variants that distinguisheach variant from the other variants to minimize cross-reactivity, andmost importantly that distinguish the variants from the wild-typehemoglobin A0. In embodiments of the invention requiring the use of avalue for the concentration of total hemoglobin in the sample, theconcentration can be determined either by an immunoassay method or anon-immunoassay method. An example of a non-immunoassay method is thedetermination of optical density. Other examples will be readilyapparent to those skilled in the hemoglobin art. In embodiments wheretotal hemoglobin is determined by immunoassay, the determination can beperformed as part of the multiplex assay. The antibody for totalhemoglobin in the multiplex assay can be either a monoclonal antibody ora polyclonal antibody, and the antibody for HbA_(1c) can be either apolyclonal antibody or a monoclonal antibody, preferably a monoclonalantibody.

Antibodies

As used herein, an “antibody” refers to a protein functionally definedas a binding protein and structurally defined as comprising an aminoacid sequence that is recognized by one of skill as being derived fromthe framework region of an immunoglobulin-encoding gene of an animalthat produces antibodies. An antibody can consist of one or morepolypeptides substantially encoded by immunoglobulin genes or fragmentsof immunoglobulin genes. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon and mu constant regiongenes, as well as myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains, respectively.

The term antibody as used herein includes antibody fragments that retainbinding specificity. For example, there are a number of wellcharacterized antibody fragments. Thus, for example, pepsin digests anantibody C-terminal to the disulfide linkages in the hinge region toproduce F(ab)′2, a dimer of Fab which itself is a light chain joined toVH-CH1 (Fd) by a disulfide bond. The F(ab)′2 may be reduced under mildconditions to break the disulfide linkage in the hinge region therebyconverting the (Fab′)2 dimer into an Fab′ monomer. The Fab′ monomer isessentially a Fab with all or part of the hinge region (see, FundamentalImmunology, W. E. Paul, ed., Raven Press, N.Y. (1993), for a moredetailed description of other antibody fragments). While variousantibody fragments are defined in terms of the digestion of an intactantibody, one of skill will appreciate that fragments can be synthesizedde novo either chemically or by utilizing recombinant DNA methodology.Thus, the term “antibody” also includes antibody fragments producedeither by the modification of whole antibodies or synthesized usingrecombinant DNA methodologies. Antibodies include dimers such asV_(H)-V_(L) dimers, V_(H) dimers, or V_(L) dimers, including singlechain antibodies. Alternatively, the antibody can be another fragment,such as a disulfide-stabilized Fv (dsFv). Other fragments can also begenerated using known techniques, including using recombinanttechniques. In some embodiments, antibodies include those that have beendisplayed on phage or generated by recombinant technology using vectorswhere the chains are secreted as soluble proteins, e.g., scFv, Fv, Fab,(Fab′)2 or generated by recombinant technology using vectors where thechains are secreted as soluble proteins.

As used here, an “immunological binding member having selective bindingaffinity” for an antigen, e.g., a hemoglobin variant, is typically anantibody. In some embodiments, a binding member having selective bindingaffinity for an antigen may be a peptide, e.g., that can be identifiedby screening peptide libraries, that has a selective binding interactionwith the antigen.

In one aspect, the invention provides monoclonal antibodies that bind toHb A_(1c) as well as monoclonal antibodies that specifically bind tohemoglobin variants HbS, HbC, HbE, and HbD. The sequence of hemoglobinbeta chain is as follows (SEQ ID NO:22):

VHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYHThe positions of amino acid residues in the hemoglobin beta chainreferred to herein is with reference to this amino acid sequence unlessotherwise specified.

Hb A_(1c) is glycated at the N-terminal valine. The most prevalentbeta-chain point mutations are HbS (Glu 6→Val); HbC (Glu 6→Lys); HbE(Glu 26→Lys) and HbD (Glu 121→Gln). The Glu 6, Glu 26, and Glu 121positions are indicated in the beta chain sequence by underline.

HbA₂ and HbF can also be determined in the assay of the presentinvention. Hemoglobin A₂ has two alpha chains and two delta chains; andhemoglobin F has two alpha and two gamma chains.

In the context of this invention, the term “specifically binds” or“specifically (or selectively) immunoreactive with,” or “having aselective affinity for” refers to a binding reaction where the antibodybinds to the antigen of interest. In the context of this invention, theantibody binds to the antigen of interest, e.g., HbS, including theglycated form of HbS, with an affinity that is at least 100-fold betterthan its affinity for other antigens, e.g., other hemoglobin variantssuch as HbA₀ or HbC.

“Reactivity” as used herein refers to the relative binding signal fromthe reactions of an antibody with the antigen to which it specificallybinds versus other antigens, such as other hemoglobin variants and orwild-type HbA₀. Reactivity is assessed using appropriate buffers thatpermit the antigen and antibody to bind. Reactivity can be determined,e.g., using a direct or sandwich ELISA assay. For example, a directformat assay for determining reactivity with wildtype hemoglobin and/orhemoglobin variants, can be used in which the antigen is directly boundto the ELISA plate, and the various antibodies are added to see whichones bind, followed by interrogation using a labeled anti-mouse antibodysuch as a phycoerythrin-labeled antibody. In the sandwich format, themonoclonal antibody is bound to the bead, followed by addition ofantigen, followed by interrogation with phycoerythrin-labeled universaldetection antibody, e.g., a phycoerythrin-labeled universal detectionantibody, that binds all hemoglobin species. Thus, in an example usingthe sandwich format, reactivity can be defined as the relativefluorescent signal produced when the specific antigen, e.g., an HbShemoglobin variant, is bound versus another antigen, e.g., a wildtypehemoglobin. An antibody is considered to be specific for an antigen ifit exhibits a 2-fold, typically at least a 3- or 4-fold increase, inreactivity for the reference antigen compared to another antigen that istested.

“Epitope” or “antigenic determinant” refers to a site on an antigen towhich an antibody binds. Epitopes can be formed both from contiguousamino acids or noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation. Methods ofepitope mapping are well known in the art (see, e.g., Epitope MappingProtocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed(1996)). A “minimal” epitope in the current invention is typicallydetermined by measuring binding of the antibody to overlapping peptidescovering the entire amino acid sequence of beta or alpha globin andidentifying the amino acid sequence shared by all bound peptides.Important amino acids in the “minimal” epitope are typically identifiedby alanine scanning.

As understood in the art, a “minimal” epitope may include substitutions,e.g., at positions that are not important for binding, e.g., asdetermined using alanine scanning. Such substitutions includeconservative substitutions where the alteration results in thesubstitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. The following are examples from amongthe twenty common naturally occurring amino acids of amino acids thatmay be substituted for one another: alanine and glycine; aspartic acidand glutamic acid; asparagine and glutamine; arginine and lysine; serineand threonine. Other conservative substitutions include substitutions ofone amino acid in the following group with another amino acid in thegroup: isoleucine, leucine, methionine, and valine. Phenylalanine,tyrosine, and tryptophan are also examples of residues that may besubstituted for one another.

Table 1 provides examples of immunogens utilized to generate specificmonoclonal antibodies to hemoglobin and hemoglobin variants.

TABLE 1 Examples of Peptide Immunogens Peptide SEQ ID Hemoglobin targetName sequence NO: Hemoglobin and H1 H2N-VHLTPEEKSAVTALW-C-CONH2 23variants H2 H2N-VHLTPEEASASTASW-C-CONH2 24 H2bisH2N-VHLTPEEKSASTASW-C-CONH2 25 HbS H3 H2N-VHLTPVEKSAVTALW-C-CONH2 26 HbCH4 H2N-VHLTPKEKSAVTALW-C-CONH2 27 HbE H5 H2N-CYG-NVDEVGGKALGRLLV-CONH228 H5bis H2N-CYG-VTALWGKVNVDEVGGK-CONH2 29 H10 H2N-C-Hx-EVGGKALG-CONH230 H10bis H2N-EVGGKALG-Hx-C-CONH2 31 HbD H6H2N-CYG-VLAHHFGKQFTPPVQAA-CONH2 32 H6bis H2N-QFTPPVQAAYQKVVAGV-GYC-CONH233 H9 H2N-GKQFTGKQFTGKQFT-GYC-CONH2 34 H11 H2N-C-Hx-HFGKQFTP-CONH2 35H11bis H2N-HFGKQFTP-Hx-C-CONH2 36 HbA1_(c) GP1Glucose-HN-VHLTPEE-Hx-C-CONH2 37 GP3 1-deoxyfructopyranosyl-HN-VHLTPEE-38 Hx-C-CONH2 Glycated H2 Glucose-HN-VHLTPEEASASTASW-C-CONH2 39

Table 2 provides examples of immunization regimens utilized to generatespecific monoclonal antibodies to hemoglobin and hemoglobin variants.

TABLE 2 Examples of immunization regimens Injection HbA and sequence HbSHbC HbE HbD HbA1c variants 1 native HbS H4-KLH H5bis-KLH denatured HbD +GP3-KLH native HbA0 antigen H6-KLH 2 H3-KLH H4-KLH H5bis-KLH denaturedHbD + GP3-KLH H1-KLH H6-KLH 3 H3-KLH H4-KLH H5bis-KLH denatured HbD +GP3-KLH native HbA0 H6-KLH 4 denatured HbS + denatured HbC H5bis-KLHdenatured HbD + GP3-KLH H1-KLH H3-KLH H6-KLH 5 denatured HbS + H4-KLHdenatured HbE denatured HbD + H3-KLH H6-KLH 6 denatured HbS + H4-KLHdenatured HbE denatured HbD H3-KLH 7 denatured HbE denatured HbD +H6-KLH 8 H5bis-KLH denatured HbD + H6-KLH 9 H5bis-KLH denatured HbD +H6-KLH 10 denatured HbE + denatured HbD + H5bis-KLH H6-KLH 11 denaturedHbE + H5bis-KLH 12 Subcutaneous Subcutaneous denatured HbE +Subcutaneous Intraperitoneal Subcutaneous Route of injection And andH5bis-KLH and and intraperitoneal intraperitoneal Subcutaneousintraperitoneal intraperitoneal and intraperitoneal

HbS Antibodies

Hemoglobin variant HbS has a point mutation in which glutamic acid atposition 6 of the hemoglobin beta chain is mutated to a valine.

Anti-HbS antibodies of the invention that are selective for HbS have thefollowing binding characteristics: the antibody bind to HbS with anaffinity that is at least 100-fold lower (i.e., better) than itsaffinity for HbC and HbA0. In some embodiments, the antibody binds tothe minimal HbS epitope ⁵PVEKSAVT¹² (SEQ ID NO:40). Such an antibody mayhave a reactivity in which the reactivity is such that the valine atposition 6 can be replaced by an isoleucine, but replacement with otheramino acids at that position results in a 2-fold, often a three-fold orgreater decrease in reactivity. In some embodiments, ³LTP⁵, ⁷E, and ¹⁰Aare also important for binding.

In some embodiments, the antibody binds to a minimal epitope³LTPVEKSAVT¹² (SEQ ID NO:3). In some embodiments, the antibody may havea reactivity where the valine at position 6 can be replaced by anisoleucine or alanine, but substitution with other amino acids at thatpositions results in a two-fold, often a three-fold or greater decreasein reactivity. In some embodiments, ²HLTPVEK⁸ (SEQ ID NO:41) and ¹⁰A arealso important for binding.

In some embodiments, an antibody that binds to an HbS minimal epitope,e.g., ⁵PVEKSAVT¹² (SEQ ID NO:40). may bind to variants of the HbSminimal epitope that have the valine at position 6, such as a minimalepitope comprising ⁵PVEX₂X₃A¹⁰ (SEQ ID NO:4), where X₂ and X₃ can beindependently selected from the 20 common naturally occurring aminoacids, e.g., conservative substitutions of K and S, respectively.

The antibody typically is an IgG, e.g., the antibody may have an IgG1,IgG2, or IgG3 isotype. In some embodiments, the light chain constantregion is a kappa chain. In other embodiments, the light chain constantregion may be a lambda chain.

In one embodiment, an HbS antibody of the invention is raised againstthe immunogen HbS and H3-KLH: H₂N-VHLTPVEKSAVTALW-C—CONH₂ (SEQ IDNO:26). In other embodiments, the immunogen is either a combination ofH3-KLH: H₂N-VHLTPVEKSAVTALW-C—CONH₂ (SEQ ID NO:26) and purified nativeand/or denatured HbS protein, or sequential or serial immunizationsusing the individual components of the above immunogens. Carrierproteins other than KLH can also be used. Examples are albumin andovalbumin, and further examples will be readily apparent to thoseskilled in the art.

As understood in the art and illustrated by Table 1 above, manyvariations of immunogens can be used to obtain the desired antibody. Forexample, peptide immunogen H3-KLH: H2N-VHLTPVEKSAVTALW-C—CONH₂ (SEQ IDNO:26) may also have a C-terminal carboxylate, rather than a C-terminalcarboxamide. In some embodiments, the cysteine linker moiety may bespaced with a Hx residue, which is 6-amino hexanoic acid, or a spacer,such as a Gly-Gly spacer sequence may be employed. Further, the peptidesequence may also vary.

An anti-HbS antibody typically binds to both glycated and nonglycatedforms of HbS with similar affinity. For example, an anti-HbS antibodytypically selectively binds to both glycated and non-glycated HbS with abinding reactivity in which there is less than a three-fold reactivitydifference, typically less than a two-fold reactivity difference,between binding to glycated vs. non-glycated HbS.

Anti-HbC Antibodies

Hemoglobin variant HbC has a lysine substituted for the glutamic acid atposition 6 of the hemoglobin beta chain. An anti-HbC monoclonal antibodyfor use in the invention typically binds to HbC with an affinity that isat least 100 times greater that the affinity of the antibody for HbS andHbA0. In some embodiments, the monoclonal antibody binds to the minimalepitope ⁴TPKEKSAVT¹² (SEQ ID NO:1). In some embodiments, the antibodyhas a binding specificity such that residues important for binding areresidues ³LT⁴ and ⁶K. In some embodiments, residues important forbinding may be ³LT⁴ and ⁶KE⁷. The binding specificity also allows forsubstitution of lysine by arginine or histidine at position 6, butsubstitution of other amino acids results in at least a 2-fold,typically a 3-fold or greater loss in reactivity. In other embodiments,the reactivity of the HbC antibody is such that the lysine at position 6may be substituted with an arginine, tyrosine, asparagine, glutamine orglycine, but substitution with other amino acids residues results in aloss of reactivity.

In some embodiments, an antibody that binds to a HbC minimal epitope,e.g., ⁴TPKEKSAVT¹² (SEQ ID NO:1), may bind to variants of the HbCminimal epitope that have the K at position 6, such as a minimal epitopecomprising ⁴TX₁KE⁷ or ³LTX₁KE⁷ (SEQ ID NO:2) where X₁ can be one of the20 common naturally occurring amino acids.

The antibody typically is an IgG, e.g., the antibody may have an IgG1,IgG2, or IgG3 isotype. In some embodiments, the light chain constantregion is a kappa chain. In other embodiments, the light chain constantregion may be a lambda chain.

An antibody of the invention may be raised against the immunogen H4-KLH:H2N-VHLTPKEKSAVTALW-C—CONH₂ (SEQ ID NO:27). Examples of other peptideimmunogens are listed in Table 1, and here again, other common carrierproteins can be used in place of KLH. In some embodiments, theimmunization is performed using a combination of the peptide andpurified native and/or denatured HbC protein. In some embodiments,sequential or serial immunizations are performed using the individualcomponents of the above immunogens. An exemplary immunization protocolis shown in Table 2. As explained above in the section relating toanti-HbS antibodies, one of skill can readily design other immunogenicpeptides to obtain an antibody having the desired HbC bindingspecificity.

An anti-HbC antibody typically binds to both glycated and nonglycatedforms of HbC with similar affinity. For example, an anti-HbC antibodytypically selectively binds to both glycated and non-glycated HbC with abinding reactivity in which there is less than a three-fold reactivitydifference, typically less than a two-fold reactivity difference,between binding to glycated vs. non-glycated HbC.

Anti-HbE Antibodies

HbE has a lysine substituted for the glutamic acid at position 26 of thehemoglobin beta chain. An anti-HbE monoclonal antibody of the inventionis typically at least 4-fold or 5-fold more reactive, often at least10-fold more reactive, with HbE in comparison to HbA. In someembodiments, the monoclonal antibody binds to the minimal epitope²²EVGGK²⁶ (SEQ ID NO:6). In some embodiments such an anti-Hb-E antibodyhas a binding specificity for ²²EVGGK²⁶ (SEQ ID NO:6) that is dependenton ²²E and in which ²¹D, ²³V, and ²⁶K are important for binding. In someembodiments, the antibody has a binding specificity that is dependent onE22 and in which D21, V23 and K26 are important for binding. In someembodiments, the antibody has a binding specificity such thatsubstitution of the K at position 26 with S, T A, R, Q or G preserves atleast 50%, typically at least 70% or greater of the binding activity. Insome embodiments, substitution of the K at position 26 with S, T, A, Ror V preserves at least 50%, typically at least 70% or greater, of thebinding activity.

In some embodiments, an antibody that binds to a HbE minimal epitope,e.g., ²²EVGGK²⁶ (SEQ ID NO:6) may bind to variants of the HbE minimalepitope that have the K at position 26, such as a minimal epitopecomprising ²¹DEVGGK²⁶ (SEQ ID NO:7) or ²²EVX₄X₅K²⁶, where X₄ and X₅ canbe independently selected from the 20 common naturally occurring aminoacids, e.g., conservative substitutions of G.

The antibody typically is an IgG, e.g., the antibody may have an IgG1,IgG2, or IgG3 isotype. In some embodiments, the light chain constantregion is a kappa chain. In other embodiments, the light chain constantregion may be a lambda chain.

An anti-HbE antibody of the invention can be obtained, e.g., using theimmunogen H5bis-KLH: H2N-CYG-VTALWGKVNVDEVGGK-CONH₂ (SEQ ID NO:29). Insome embodiments, the antibody is raised against an immunogen H5bis-KLH:H2N-CYG-VTALWGKVNVDEVGGK-CONH₂ (SEQ ID NO:29) with mixtures orsequential injections of peptide, native HbE antigen, and HbE denaturedantigen. Examples of peptide immunogens are provided in Table 1. Peptideimmunogens can be used in combination with one another, either with orwithout denatured or native HbE. Exemplary immunization protocols areprovided in Table 2. As explained above in the section relating toanti-HbS antibodies, one of skill can readily design other immunogenicpeptides to obtain an antibody having the desired HbE bindingspecificity. The reader is again referred to Table 1 for examples ofother peptide immunogens.

An anti-HbE antibody typically binds to both glycated and nonglycatedforms of HbE with similar affinity. For example, an anti-HbE antibodytypically selectively binds to both glycated and non-glycated HbE with abinding reactivity in which there is less than a three-fold reactivitydifference, typically less than a two-fold reactivity difference,between binding to glycated vs. non-glycated HbE.

Anti-HbD Antibodies

HbD has a glutamine substituted for a glutamic acid at position 121 ofthe hemoglobin beta chain. An anti-HbD monoclonal antibody of theinvention is typically at least 3-fold, or greater more reactive withHbD in comparison to HbA. In some embodiments, the antibody binds to theminimal epitope ¹²¹QFTPP¹²⁵ (SEQ ID NO:8). In some embodiments, theantibody has a binding specificity where residues ¹¹⁹G, ¹²¹QF¹²², and¹²⁴PP¹²⁵ are important for binding.

In some embodiments, an antibody that binds to a HbD minimal epitope,e.g., ¹²¹QFTPP¹²⁵ (SEQ ID NO:8), may bind to variants of the HbE minimalepitope that have the Q at position 121, such as a minimal epitopecomprising ¹¹⁹GX₆QFX₇PP¹²⁵ (SEQ ID NO:9) or ¹²¹QFX₇PP¹²⁵ (SEQ ID NO:10),where X₆ and X₇ can be independently selected from the 20 commonnaturally occurring amino acids, e.g., conservative substitutions of Kand T, respectively.

The antibody typically is an IgG, e.g., the antibody may have an IgG1 orIgG2 isotype. In some embodiments, the light chain constant region is akappa chain. In other embodiments, the light chain constant region maybe a lambda chain.

An anti-HbD antibody of the invention can be raised, for example,against the immunogen H6-KLH: H2N-CYGVLAHHFGKQFTPPVQAA-CONH₂ (SEQ IDNO:32), or against mixtures of native and/or denatured HbD and H6-KLH:H2N-CYGVLAHHFGKQFTPPVQAA-CONH₂ (SEQ ID NO:32), or by using combinationsof, or sequential injections of, the various immunogens. Otherimmunogenic peptides useful in obtaining an antibody having the desiredHbD binding specificity will be readily apparent to those skilled in theart.

An anti-HbD antibody typically binds to both glycated and nonglycatedforms of HbD with similar affinity. For example, an anti-HbD antibodytypically selectively binds to both glycated and non-glycated HbD with abinding reactivity in which there is less than a three-fold reactivitydifference, typically less than a two-fold reactivity difference,between binding to glycated vs. non-glycated HbD.

Pan-Reactive Antibodies

The invention also provides pan-reactive antibodies for use in theinvention. Such antibodies bind to multiple forms of hemoglobin.Pan-reactive antibodies can be produced using a number of differentimmunogens, including H5bis-KLH: H2N-CYGVTALWGKVNVDEVGGK-CONH₂ (SEQ IDNO:29) or H1-KLH: H2N-VHLTPEEKSAVTALW-C—CONH₂ (SEQ ID NO:23). Suchpeptide immunogens can be injected either in a mixture with native ordenatured HbA₀, or sequentially with native and/or denatured HbA0. Asunderstood in the art, any number of Hb immunogens can be used to obtaina Hb antibody that selectively binds to HbA₀ as well as Hb variants.Pan-reactive antibodies may be monoclonal or polyclonal. Pan-reactiveantibodies can also be obtained by immunization with native or denaturedhemoglobin without using peptide immunogens.

In some embodiments, a pan-reactive polyclonal antibody for use in theinvention binds to one ore more epitopes present in the followingregions of alpha globin and beta globin: alpha globin⁴⁹SHGSAQVKGHGKKVADALTNAVAHVDDMPNALSALSDHLHAHKLRRVDPV⁹⁶ (SEQ ID NO:11),beta globin ¹⁵WGKVNVDEVGGEALG²⁹ (SEQ ID NO:12), ⁴⁵FGDLSTP⁵¹ (SEQ IDNO:13), and ⁷⁶AHLDNLKGTFAT⁸⁷ (SEQ ID NO:14).

In one embodiment, a pan-reactive antibody binds to the beta globinepitope ⁹SAVTALWGKVNV²⁰ (SEQ ID NO:15). In some embodiments, theantibody binds to the beta globin epitope ¹¹VTALW¹⁵ (SEQ ID NO:17). Insome embodiments ¹¹VT¹² and ¹⁴LW¹⁵ are important for binding.

In some embodiments, a pan-reactive antibody binds to beta and alphaglobin epitopes that contain the following sequences: a beta globinminimal epitope ⁸KSAVTALWGKVNV²⁰ (SEQ ID NO:16), a beta globin minimalepitope ⁵⁸PKVKAHGKKVLGAF⁷¹ (SEQ ID NO:20) and a beta globin minimalepitope ⁸⁷TLSELHCDKLHVDPENFR¹⁰⁴ (SEQ ID NO:21). In some embodimentsresidues ¹³ALwG¹⁶ (SEQ ID NO:18) are important for binding.

The antibody typically is an IgG, e.g., the antibody may have an IgG1,IgG2, or IgG3 isotype. In some embodiments, the light chain constantregion is a kappa chain. In other embodiments, the light chain constantregion may be a lambda chain.

A pan-reactive antibody used in the invention is broadly reactive tohemoglobin and binds to both glycated and non-glycated forms ofhemoglobin A and variants such as HbS, HbC, HbD, and HbE.

In some embodiments of the invention, a pan-reactive antibody that bindsto multiple forms of hemoglobin is used as a labeled binding member thatbinds to all of the analytes, thereby labeling the bound analytes. Thus,for example, a labeled pan-reactive polyclonal antibody that binds tothe epitopes: alpha globin⁴⁹SHGSAQVKGHGKKVADALTNAVAHVDDMPNALSALSDHLHAHKLRRVDPV⁹⁶ SEQ ID NO:11),beta globin ¹⁶GKVNVDEVGGEALG²⁹ SEQ ID NO:42), ⁴⁶GDLSTP⁵¹ SEQ ID NO:43),and ⁷⁸LDNLKGTFAT⁸⁷ (SEQ ID NO:44) can be used as a universal detectionantibody that binds to all of the analytes (forms of hemoglobin) beingassayed, thereby labeling the bound analytes

Anti-HbA_(1c) Antibodies

The invention additionally provides anti-HbA_(1c) antibodies that have abinding specificity for glycated hemoglobin. Such antibodies can beproduced using an immunogen such as GP3-KLH:1-deoxyfructopyranosyl-HN-VHLTPEE-Hx-C—CONH₂ (SEQ ID NO:38). Theantibody can be an IgG, for example, and can have an IgG₁, IgG₂, or IgG₃isotype. In some embodiments, the light chain constant region is alambda chain. In other embodiments, the light chain constant region is akappa chain.

An anti-HbA_(1c) antibody of the invention is highly specific forglycated hemoglobin, including HbA_(1c), HbS_(1c), HbD_(1c), HbE_(1c),and HbC_(1c), and does not recognize non-glycated forms of hemoglobin(i.e., the antibody has at least a 100-fold greater affinity forHbA_(1c), HbS_(1c), HbD_(1c), HbE_(1c), and HbC_(1c) than for thenon-glycated forms). Such antibodies typically have a bindingspecificity for glycated N-terminal peptide where both glycated valine 1and histidine 2 are important residues for binding.

An A_(1c) monoclonal antibody has a binding specificity in competitivebinding experiments such that the glycated peptide GP3(1-deoxyfructopyranosyl-HN-VHLTPEE-Hx-C—CONH₂; SEQ ID NO:38) competesfor binding to native HbA_(1c), but unglycated peptides such as RW1a(VHLTPEE-CONH₂; SEQ ID NO:45) do not.

HbF and HbA₂ Antibodies

HbF and HbA₂ can also be assayed using the methods of the invention.Antibodies that selectively bind to HbF relative to HbA₀ or other Hbproteins; or to HbA₂ relative to HbA₀ or other Hb proteins, can beobtained using immunogens comprising peptide sequences that are specificto HbF or sequences that are specific to HbA₂, as there are multipledifferences in the delta and gamma chains relative to the A₀ beta chain.

Generation of Antibodies

The anti-hemoglobin antibodies of the invention can be raised againsthemoglobin proteins, or fragments, or produced recombinantly. Any numberof techniques well known in the art can be used to determine antibodybinding specificity. See, e.g., Harlow & Lane, Antibodies, A LaboratoryManual (1988) for a description of immunoassay formats and conditionsthat can be used to determine specific immunoreactivity of an antibody

In some embodiments, an antibody for use in the invention, e.g., ahemoglobin antibody that binds various forms of hemoglobin, a hemoglobinantibody specific for a variant, or a hemoglobin antibody specific forglycated hemoglobin, is a polyclonal antibody. For example, an antibodyspecific for a hemoglobin variant can be an affinity-purifiedmonospecific polyclonal antibody. Methods of preparing polyclonalantibodies are known to the skilled artisan (e.g., Harlow & Lane,Antibodies, A Laboratory manual (1988); Methods in Immunology).Polyclonal antibodies can be raised in a mammal by one or moreinjections of an immunizing agent and, if desired, an adjuvant.

In some embodiments, the antibody for use in the invention, e.g., anantibody that binds to multiple forms of hemoglobin, an antibody that isspecific for a hemoglobin variant (and the glycated hemoglobin variant),or an antibody that is specific for glycated hemoglobin, is a monoclonalantibody. Monoclonal antibodies may be prepared using hybridoma methods,such as those described by Kohler & Milstein, Nature 256:495 (1975). Ina hybridoma method, a mouse, rat, rabbit, or other appropriate hostanimal, is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

As stated above, antibodies of the invention can be generated using anynumber of immunogens and immunization protocols. In some embodiments,the immunogen is a peptide that is administered in combination with anative or denatured hemoglobin protein. As understood in the art, animmunogen may be administered multiple times. In embodiments in which acombination is employed, the combination of antigens may be administeredconcurrently, or sequentially, in any order. In some embodiments, apeptide immunogen is a KLH conjugate, however, carrier proteins otherthan KLH can be used, e.g., BSA conjugates can be used.

Assay Conditions Using the Antibodies

In the practice of this invention, hemoglobin A_(1c), hemoglobinvariants, and total hemoglobin can be measured via a variety ofimmunoassay formats. One example is the sandwich format, in which aspecific antibody to the analyte is attached to the solid phase bead,and detection is accomplished by interrogating the beads with one ormore antibodies to the hemoglobin species. A universal antibody whichbinds to all hemoglobin species can be used for the interrogation, buttwo or more detection antibodies can also be used. An example is shownin FIG. 1, where individual antibodies are used that are specific tototal hemoglobin, HbA_(1c), and the four most prevalent hemoglobinvariation species HbS, HbC, HbE, and HbD, respectively, are bound toseparate subpopulations of beads, while a universal antibody that bindsto all hemoglobin species and that bears phycoerythrin (PE) as a labelis used. In a sandwich assay format, the quantity of antibody for eachassay is selected such that the analyte (the particular form ofhemoglobin to which the assay is directed) is in excess, so that theantibodies are the limiting reagents in the binding reactions.Competition between the antibody for total hemoglobin and the antibodiesfor the individual hemoglobins, for example, is thereby minimized. Byadjusting the assay parameters and selection of the appropriateantibodies in a manner within the skill of the art, however, acompetitive assay format can also be utilized for multiplexed detectionof hemoglobin species.

While the multiplex assay can be utilized on mammalian blood samples ingeneral, the assay is of particular value to samples of human blood.Blood samples are prepared for the assay by lysis of the cells anddilution of the lysate to a concentration suitable for immunoassay. Eachof these steps is performed by methods known in the art. Dilution can beachieved with water, solutions containing saponin, or any other diluentthat will not affect the hemoglobins or their immunological bindingactivity, and the degree of dilution can vary widely. In most cases, thedilution will be within the range of about 1:25 to about 1:3000. Thehemoglobins in the lysate can be denatured before or after dilution ofthe lysate and used in the assay, or the lysate can be used withoutdenaturation of the hemoglobins. In most cases, denaturation ispreferred, and can be performed by methods known in the art.

The levels of HbA_(1c) and each of the variants are preferably eachexpressed as a percentage of total hemoglobin in the sample. Fordeterminations of degrees of glycation in the presence of a hemoglobinvariant, the invention offers three options. One option, which iscompatible with the currently accepted method, is the determination ofthe HbA_(1c) level by the result from the HbA_(1c) bead only, normalizedto total hemoglobin. The second option is the determination of the totalhemoglobin glycation by adding the percent of the glycated form of thevariant to the percent HbA_(1c). The third option, which is useful inthe event that the determination of HbA_(1c) is adversely affected bythe presence of the variant, is to adjust the as-measured percentHbA_(1c) by a correction factor that is a function of the detected levelof the variant. The function can be determined empirically by a relationthat can be independently determined by separate assays, includingnon-multiplex assays. The correction factor can be one that is appliedeither to the HbA_(1c) concentration after the concentration has beennormalized with respect to total hemoglobin, or to the concentrationprior to normalization.

To illustrate the correction of the HbA_(1c) value, assays wereperformed on samples from ten patients, using both a bead-based assay(BioPlex 2200 of Bio-Rad Laboratories, Inc., Hercules, Calif., USA) inaccordance with the present invention and an HPLC assay (Variant™ II ofBio-Rad Laboratories, Inc.), both assays determining percent HbA_(1c) asa function of increasing percentage of HbC. The results are shown inTable 3 and in FIG. 2, in which the “Difference Ratio”=(% A_(1c) VariantII−% A_(1c) BioPlex 2200)/(% A_(1c) Variant II).

TABLE 3 % A_(1c) Variant Adjusted Patient BioPlex II Difference BioPlexDifference from Target ID % HbC 2200 (Target) Ratio Value AdjustedUnadjusted PT 200 31.8 6.06 5.9 −0.03 5.73 0.17 −0.16 PT 219 32.1 6.305.8 −0.09 5.98 −0.18 −0.50 PT 265 33.7 5.71 5.4 −0.06 5.37 0.03 −0.31 PT622 34.1 6.20 5.9 −0.05 5.87 0.03 −0.30 PT 658 33.9 5.92 5.9 0.00 5.590.31 −0.02 PT 667 33.5 6.00 5.8 −0.03 5.67 0.13 −0.20 PT 32.1 6.14 6.1−0.01 5.81 0.29 −0.04 m832 PT 37.6 5.80 5.5 −0.05 5.47 0.03 −0.30 m837PT 39.2 5.37 4.6 −0.17 5.03 −0.43 −0.77 m908 PT 41.1 5.53 4.8 −0.15 5.19−0.39 −0.73 m923 average difference → 0.00 −0.33

While various mathematical models can be used to quantify therelationship of the percent HbA_(1c) difference as a function of percenthemoglobin C (or any hemoglobin variant), the mathematical model used inthis example is a simple linear regression model. Using this model, thevalues obtained from the BioPlex 2200 immunoassay can be corrected toyield a result comparable to the reference method. This is demonstratedby the average difference of the adjusted BioPlex 2200 percent HbA_(1c)value relative to the target percent HbA_(1c) value determined by thereference Variant II method. In Table 3 and FIG. 2, the averagedifference is zero for the adjusted HbA_(1c) values compared to −0.33for the corresponding unadjusted values. The corrected HbA_(1c) valueshown in the table provides a better estimate of the glycemic index ofthe individual.

The BioPlex 2200 bead-based immunoassay used in the obtaining the datain Table 3 and FIG. 2 utilizes an antibody that binds HbA_(1c) and allhemoglobin variants including HbS, HbC, HbD, and HbE. All glycatedvariants and HbA₀ are bound with approximately the same affinity andavidity by the antibody. The immunoassay result thus represents totalglycated hemoglobin, which in the case of a heterozygous hemoglobin ASvariant, is the combined value that includes both the HbA_(1c) andHbS_(1c) species. In individuals exhibiting a hemoglobin variantphenotype, the proportion of the glycated hemoglobin corresponding tothe variant provides an improved measure of glycemic status. The percentHbS_(1c) in the sample is obtained by multiplying the total percentHbA_(1c) plus HbS_(1c) value by the proportion of HbS in the sample. Forexample, for a patient sample with a total glycated hemoglobin value of5.54% (consisting of HbA_(1c) and HbS_(1c)), multiplying this value bythe proportion of HbS in the sample of 38.8% yields a value for HbS_(1c)of 2.14%. The remainder of the glycated material is HbA_(1c) at 3.4%.This again is but one mathematical model; more sophisticatedmathematical models can be used to provide more accurate results asneeded.

The beads that provide the surfaces on which the binding reactions occurin the practice of this invention can be formed of any material that isinert to the assay materials and to the components of the sample itself,and that is solid and insoluble in the sample and in any other solventsor carriers used in the assay. Polymers are preferred, and the beads arepreferably microparticles. The polymeric can be any material that can beformed into a microparticle and is capable of coupling to an antibody ata region on the antibody that does not interfere with theantigen-binding regions of the antibody. In embodiments in whichfluorescent labels are used, preferred polymers are also those thatproduce at most a minimal level of autofluorescence. Examples ofsuitable polymers are polyesters, polyethers, polyolefins, polyalkyleneoxides, polyamides, polyurethanes, polysaccharides, celluloses, andpolyisoprenes. Crosslinking is useful in many polymers for impartingstructural integrity and rigidity to the microparticle. Magnetic beadscan also be used.

Attachment of the antibodies to the surfaces of the beads can beachieved by electrostatic attraction, specific affinity interaction,hydrophobic interaction, or covalent bonding. Covalent bonding ispreferred. Functional groups for covalent bonding can be incorporatedinto the polymer structure by conventional means, such as the use ofmonomers that contain the functional groups, either as the sole monomeror as a co-monomer. Examples of suitable functional groups are aminegroups (—NH₂), ammonium groups (—NH₃ ⁺ or —NR₃ ⁺), hydroxyl groups(—OH), carboxylic acid groups (—COOH), and isocyanate groups (—NCO).Useful monomers for introducing carboxylic acid groups into polyolefins,for example, are acrylic acid and methacrylic acid. Linking groups canalso be used for increasing the density of the antibodies on the solidphase surface and for decreasing steric hindrance to increase the rangeand sensitivity of the assay. Examples of suitable useful linking groupsare polylysine, polyaspartic acid, polyglutamic acid and polyarginine.

The size range of the beads can vary and particular size ranges are notcritical to the invention. In most cases, the aggregated size range ofthe beads lies within the range of from about 0.3 micrometers to about100 micrometers in diameter, and preferably within the range of fromabout 0.5 micrometers to about 40 micrometers.

Multiplexing with the use of beads in accordance with this invention isachieved by assigning the beads to two or more groups, also referred toherein as bead sets or subpopulations.

Each group will have affixed thereto an antibody selected for either ahemoglobin variant, a glycated variant, HbA_(1c), or total hemoglobin,and will be separable or at least distinguishable from the othergroup(s) by a “differentiation parameter.” The “differentiationparameter” can be any distinguishable characteristic that permitsseparate detection of the assay result in one group from those in theother groups. One example of a differentiation parameter is the particlesize, with each group having a size range that does not overlap with thesize ranges of the other groups. The widths of the size ranges and thespacing between mean diameters of different size ranges are selected topermit differentiation of the groups by flow cytometry according tosize, and will be readily apparent to those skilled in the use of andinstrumentation for flow cytometry. In this specification, the term“mean diameter” refers to a number average diameter. In most cases, apreferred size range width is one with a CV of about ±5% or less of themean diameter, where CV is the coefficient of variation and is definedas the standard deviation of the particle diameter divided by the meanparticle diameter times 100 percent. The minimum spacing between meandiameters among the various size ranges can vary depending on the sizedistribution, the ease of segregating beads by size for purposes ofattaching different antibodies, and the type and sensitivity of the flowcytometry equipment. In most cases, best results will be achieved whenthe mean diameters of different size ranges are spaced apart by at leastabout 6% of the mean diameter of one of the size ranges, preferably atleast about 8% of the mean diameter of one of the size ranges and mostpreferably at least about 10% of the mean diameter of one of the sizeranges. Another preferred size range width relation is that in which thestandard deviation of the particle diameters within each size range isless than one third of the separation of the mean diameters of adjacentsize ranges.

Another example of a differentiation parameter that can be used todistinguish among the various groups of beads is fluorescence.Differentiation by fluorescence is accomplished by incorporatingfluorescent materials in the beads, the materials having differentfluorescent emission spectra for each group of beads and beingdistinguishable on this basis.

Fluorescence can thus be used both as a differentiation parameter and asa means for detecting that binding has occurred in the assays performedon the beads. The latter can be achieved by fluorescent labels servingas assay reporters. Thus, while individual groups can be distinguishedby emitting different emission spectra, and the emission spectra usedfor group differentiation purposes can themselves differ from theemission spectra of the assay reporters. An example of a fluorescentsubstance that can be used as a differentiation parameter is fluoresceinand an example of a substance that can be used for the assay detectionis phycoerythrin. Different bead groups can be distinguished from eachother by being dyed with different concentrations of fluorescein.Different bead groups can be distinguished by using fluorescentmaterials that have different fluorescence intensities or that emitfluorescence at different wavelengths. The dyes can also be used incombinations to produce a plurality of fluorescent emissions atdifferent wavelengths, and the wavelength difference can be used both asthe differentiation parameter and as a means of distinguishing thedifferentiation parameter from the assay reporter.

Still other examples of useful differentiation parameters are lightscatter, light emission, or combinations of light scatter and emission.Side-angle light scatter varies with particle size, granularity,absorbance and surface roughness, while forward-angle light scatter ismainly affected by size and refractive index. Any of these qualities canthus be used as the differentiation parameter.

According to one means of differentiation, the beads will have two ormore fluorochromes incorporated within them so that each bead in thearray will have at least three distinguishable parameters associatedwith it, i.e., side scatter together with fluorescent emissions at twoseparate wavelengths. A red fluorochrome such as Cy5 can thus be usedtogether with an orange fluorochrome such as Cy5.5. Additionalfluorochromes can be used to expand the system further. Each bead canthus contain a plurality of fluorescent dyes at varying wavelengths.

Still another example of a differentiation parameter that can be used todistinguish among the various groups of beads is absorbance. When lightis applied to beads the absorbance of the light by the beads isindicated mostly by the strength of the laterally (side-angle) scatteredlight while the strength of the forward-scattered light is relativelyunaffected. Consequently, the difference in absorbance between variouscolored dyes associated with the beads is determined by observingdifferences in the strength of the laterally scattered light.

A still further example of a differentiation parameter that can be usedto distinguish among the various groups of beads is the number of beadsin each group. The number of beads of each group in an assay is variedin a known way, and the count of beads having various assay responses isdetermined. The various responses are associated with a particular assayby the number of beads having each response.

As the above examples illustrate, a wide array of parameters orcharacteristics can be used as differentiation parameters to distinguishthe beads of one group from those of another. The differentiationparameters may arise from size, composition, physical characteristicsthat affect light scattering, excitable fluorescent or colored dyes thatimpart different emission spectra and/or scattering characteristics tothe beads, or different concentrations of one or more fluorescent dyes.When the differentiation parameter is a fluorescent dye or color, it canbe coated on the surface of the beads, embedded in the beads, or boundto the molecules of the bead material. Thus, fluorescent beads can bemanufactured by combining the polymer material with the fluorescent dye,or by impregnating the beads with the dye. Beads with dyes alreadyincorporated and thereby suitable for use in the present invention arecommercially available, from suppliers such as Spherotech, Inc.(Libertyville, Ill., USA) and Molecular Probes, Inc. (Eugene, Oreg.,USA). A list of vendors of flow cytometric products can be found on theInternet, e.g., at the world wide webmolbio.princeton.edu/facs/FCMsites.html site.

Detection and differentiation in accordance with this invention areperformed by flow cytometry. Methods of and instrumentation for flowcytometry are known in the art, and those that are known can be used inthe practice of the present invention. Flow cytometry in general residesin the passage of a suspension of beads or microparticles as a streampast a light beam and electro-optical sensors, in such a manner thatonly one particle at a time passes through the region. As each particlepasses this region, the light beam is perturbed by the presence of theparticle, and the resulting scattered and fluorescent light aredetected. The optical signals are used by the instrumentation toidentify the subgroup to which each particle belongs, along with thepresence and amount of label, so that individual assay results areachieved. Descriptions of instrumentation and methods for flow cytometryare found in the literature. Examples are McHugh, “Flow MicrosphereImmunoassay for the Quantitative and Simultaneous Detection of MultipleSoluble Analytes,” Methods in Cell Biology 42, Part B (Academic Press,1994); McHugh et al., “Microsphere-Based Fluorescence Immunoassays UsingFlow Cytometry Instrumentation,” Clinical Flow Cytometry, Bauer, K. D.,et al., eds. (Baltimore, Md., USA: Williams and Williams, 1993), pp.535-544; Lindmo et al., “Immunometric Assay Using Mixtures of TwoParticle Types of Different Affinity,” J. Immunol. Meth. 126: 183-189(1990); McHugh, “Flow Cytometry and the Application of Microsphere-BasedFluorescence Immunoassays,” Immunochemica 5: 116 (1991); Horan et al.,“Fluid Phase Particle Fluorescence Analysis: Rheumatoid FactorSpecificity Evaluated by Laser Flow Cytophotometry,” Immunoassays in theClinical Laboratory, 185-189 (Liss 1979); Wilson et al., “A NewMicrosphere-Based Immunofluorescence Assay Using Flow Cytometry,” J.Immunol. Meth. 107: 225-230 (1988); Fulwyler et al., “Flow MicrosphereImmunoassay for the Quantitative and Simultaneous Detection of MultipleSoluble Analytes,” Meth. Cell Biol. 33: 613-629 (1990); CoulterElectronics Inc., United Kingdom Patent No. 1,561,042 (published Feb.13, 1980); and Steinkamp et al., Review of Scientific Instruments 44(9):1301-1310 (1973).

Example 1

This example presents the binding activities of six hemoglobin candidateantibodies for use in the practice of this invention. The six antibodiesare:

19E10-E7 (HbS specific) 7B3-2C3-1G10 (HbD specific) 12C8-A11 (HbCspecific) 13G7-E8-3H3 (HbA_(1c) specific) 4A10-2D6-2G8 (HbE specific)3E5-DLE10-3A3 (pan-reactive).

19E10-E7 binds to a beta globin minimal epitope ⁵PVEKSAVT¹² (SEQ IDNO:40). ⁵PVE⁷ and A¹⁰ are important for binding. L³ and T⁴ alsocontribute to binding activity. Additional epitope mapping experimentsshowed that V⁶ can be replaced with I without loss of binding activity.

12C8-A11 binds to a beta globin minimal epitope ⁴TPKEKSAVT¹² (SEQ IDNO:1). T⁴ and K⁶ are important for binding. L³ also contributes tobinding. Additional epitope mapping experiments showed that K⁶ can bereplaced with R without reducing binding activity.

4A₁₀-2D6-2G8 binds a beta globin minimal epitope ²²EVGGK²⁶ (SEQ IDNO:6). ²²EV²³ and K²⁶ are important for binding. D²¹ also contributes tobinding. Additional epitope mapping experiments showed that K²⁶ can bereplaced with S or T without loss of binding activity.

7B3-2C3-1G10 binds to a beta globin minimal eptiope ¹²¹QFTPP¹²⁵ (SEQ IDNO:8). G¹¹⁹ also contributes to binding.

The antibody binding kinetics were analyzed using the ProteOn XPR36(Bio-Rad Laboratories, Inc.) for protein-protein interactions. Thedifferent antibodies (10 μg of each) were amine-coupled to the sensorchip such that one antibody was immobilized per channel. Antigen wasemployed in the range of from 200 to 13 nM. The results of the kineticanalysis are summarized in Table 4 below.

Each antibody had a high affinity for its specific hemoglobin. Thepan-reactive antibody also had good affinity constants to the differentantigens, but lower than the affinity constant exhibited by the specificvariant antibodies for their respective antigens. Except for 19E10-E7,all of the variant antibodies did not bind HbA0, so the affinityconstants were essentially zero. For the 19E10-E7 anti-HbS antibody, alow level of binding to HbA₀ was observed, with an affinity constant of6.5 10⁻⁷ M, which is 2 logs less than the affinity constant for HbS.

TABLE 4 Kinetics analysis for 6 specific monoclonal antibodies tohemoglobin and hemoglobin variants k_(a) (1/Ms) K_(d) (1/s) K_(D) (M)19E10-E7 HbS 7.6 10⁴ 6.3 10⁻⁴ 8.3 10⁻⁹ 12C8-A11 HbC 7.3 10⁴ 4.5 10⁻⁴ 6.110⁻⁹ 4A10-2D6 HbE 7.1 10⁴ 6.2 10⁻⁵  8.7 10⁻¹⁰ 7B3-2C3 HbD 1.4 10⁵ 1.110⁻³ 7.4 10⁻⁹ 13G7-E8 HbA_(1c) 1.2 10⁴ 2.3 10⁻⁵ 1.9 10⁻⁹ 3E5-DLE10 HbS3.9 10⁴ 7.9 10⁻⁴ 2.0 10⁻⁸ HbE 2.9 10⁴ 4.4 10⁻⁴ 1.5 10⁻⁸ HbD 3.4 10⁴ 1.010⁻³ 3.0 10⁻⁸ HbA_(1c) 3.7 10⁴ 9.4 10⁻⁴ 2.5 10⁻⁸ HbC 4.5 10⁴ 7.4 10⁻⁴1.6 10⁻⁸ HbA0 1.4 10⁴ 3.4 10⁻³ 2.4 10⁻⁷

Example 2

This examples demonstrates the measurement of hemoglobin A_(1c) andhemoglobin variant proteins as percentages of total hemoglobin using asandwich immunoassay in accordance with this invention. Solid phasecapture bead immunoreagents were developed utilizing the six monoclonalantibodies specific to HbA₀, HbA_(1c), HbS, HbC, HbE, and HbD describedin Example 1.

Antibodies to each of the six target antigens were coupled covalently toparamagnetic beads. Each bead was dyed to contain a specific fluorescentsignal that was unique to each antibody, to enable subsequentdifferentiation in a flow cytometry detector. The six antibody-coupledbeads were mixed to create a multiplex bead reagent. A detectionantibody reagent was prepared using a phycoerythrin-labelled polyclonalantibody with reactivity to all hemoglobin species. A diagram of thevarious beads and the species bound to each in the assay is shown inFIG. 1.

The assay was performed by adding samples of whole blood and calibrators(5 μL) to a solution of buffered denaturant (10 μL), to expose all ofthe epitopes of the hemoglobin species present in the samples in orderto make them available for binding by the solid phase antibodies. Afterdenaturation for 10 minutes at 37 degrees, the bead reagent (250 μL) wasadded to the samples, followed by an additional incubation for 20minutes at 37 degrees. The reaction mixture was washed four times withphosphate-buffered saline containing 0.1% Tween-20 (PBST, 100 μL each)detergent to remove all of the unbound proteins from the sample, leavingthe beads with their bound hemoglobin targets. The beads werere-suspended in PBST containing phycoerythrin-labelled antibody reagent(25 μL), and incubated for 20 minutes at 37 degrees Celsius.

After washing four times with PBST (100 μL each), the beads wereresuspended in PBST and processed through a Luminex flow cytometrydetector to interrogate the beads for binding of the individualhemoglobin species present in the samples. For example, samples fromhomozygous hemoglobin AA individuals exhibited signal from the HbA1c andHbA₀ beads, and samples from heterozygous hemoglobin variant individualsexhibited signal from the HbA₀, HbA_(1c) and the specific hemoglobinvariant beads. The phycoerythrin-derived fluorescent signal of each beadwas measured for the samples and calibrators. A calibration curve wasconstructed for each hemoglobin analyte using the signal from the beadand the known dose of the respective calibrators. The concentration ofthe hemoglobin analytes in each sample was determined from theirfluorescent signal and the established dose-response of the calibrationcurve. Percent HbA_(1c) and percent hemoglobin variant, if any, in thesamples were determined by dividing the concentration of hemoglobinA_(1c) or variant hemoglobin protein by the concentration of HbA₀, eachderived from their respective bead. In the case of heterozygoushemoglobin variant-containing samples (such as HbAS, for example), thepercent A_(1c) value derived from the ratio of the A_(1c) to HbA₀concentrations was adjusted when needed using the concentration of thehemoglobin variant present in the sample, to provide a value that bestreflected the true glycemic index of the individual.

In the claims appended hereto, the term “a” or “an” is intended to mean“one or more.” The term “comprise” and variations thereof such as“comprises” and “comprising,” when preceding the recitation of a step oran element, are intended to mean that the addition of further steps orelements is optional and not excluded. All patents, patent applications,and other published reference materials cited in this specification arehereby incorporated herein by reference in their entirety for theirdisclosures of the subject matter in whose connection they are citedherein. Any discrepancy between any reference material cited herein orany prior art in general and an explicit teaching of this specificationis intended to be resolved in favor of the teaching in thisspecification. This includes any discrepancy between an art-understooddefinition of a word or phrase and a definition explicitly provided inthis specification of the same word or phrase.

What is claimed is:
 1. A monoclonal antibody that selectively binds tohemoglobin variant HbC and glycated HbC, wherein the antibody binds toHbC minimal epitope ⁴TPKEKSAVT¹² (SEQ ID NO:1) and is raised against andHbC immunogen H2N-VHLTPKEKSAVTALW-C—CONH2 (SEQ ID NO:27).
 2. Amonoclonal antibody that selectively binds to hemoglobin variant HbS andglycated HbS, wherein the antibody binds to HbS minimal epitope³LTPVEKSAVT¹² (SEQ ID NO:3) and is raised against an HbS immunogenH2N-VHLTPVEKSAVTALW-C—CONH2 (SEQ ID NO:26).
 3. An immunoassay reagentcomposition comprising a monoclonal antibody that selectively binds tohemoglobin variant HbC and glycated HbC, wherein the antibody binds toHbC minimal epitope ⁴TPKEKSAVT¹² (SEQ ID NO:1) and is raised against andHbC immunogen H2N-VHLTPKEKSAVTALW-C—CONH₂ (SEQ ID NO:27); or amonoclonal antibody that selectively binds to hemoglobin variant HbS andglycated HbS, wherein the antibody binds to HbS minimal epitope³LTPVEKSAVT¹² (SEQ ID NO:3) and is raised against an HbS immunogenH2N-VHLTPVEKSAVTALW-C—CONH₂ (SEQ ID NO:26).
 4. The immunoassay reagentcomposition of claim 3, comprising the monoclonal antibody thatselectively binds to hemoglobin variant HbC and glycated HbC and themonoclonal antibody that selectively binds to hemoglobin variant HbS. 5.The immunoassay composition of claim 3, further comprising a monoclonalantibody that selectively binds to hemoglobin variant HbD and glycatedHbD and is raised against an HbD immunogenH₂N-CYG-VLAHHFGKQFTPPVQAA-CONH₂ (SEQ ID NO:32) orH₂N-QFTPPVQAAYQKVVAGV-GYC—CONH₂ (SEQ ID NO:33); or a monoclonal antibodythat selectively binds to hemoglobin variant HbE and glycated HbE and israised against an HbE immunogen H₂N-CYG-VTALWGKVNVDEVGGK-CONH₂ (SEQ IDNO:29).
 6. The immunoassay composition of claim 3, further comprising amonoclonal antibody that selectively binds to hemoglobin variant HbD andglycated HbD and is raised against an HbD immunogenH₂N-CYG-VLAHHFGKQFTPPVQAA-CONH₂ (SEQ ID NO:32) orH₂N-QFTPPVQAAYQKVVAGV-GYC—CONH₂ (SEQ ID NO:33); and a monoclonalantibody that selectively binds to hemoglobin variant HbE and glycatedHbE and is raised against an HbE immunogenH₂N-CYG-VTALWGKVNVDEVGGK-CONH₂ (SEQ ID NO:29).
 7. The immunoassaycomposition of claim 4, further comprising a monoclonal antibody thatselectively binds to hemoglobin variant HbD and glycated HbD and israised against an HbD immunogen H₂N-CYG-VLAHHFGKQFTPPVQAA-CONH₂ (SEQ IDNO:32) or H₂N-QFTPPVQAAYQKVVAGV-GYC—CONH₂ (SEQ ID NO:33); or amonoclonal antibody that selectively binds to hemoglobin variant HbE andglycated HbE and is raised against an HbE immunogenH₂N-CYG-VTALWGKVNVDEVGGK-CONH₂ (SEQ ID NO:29).
 8. The immunoassaycomposition of claim 7, further comprising a monoclonal antibody thatselectively binds to a hemoglobin beta chain, wherein the antibody bindsto a hemoglobin beta chain minimal epitope ¹¹VTALW¹⁵ (SEQ ID NO:17) andis raised against a hemoglobin beta chain immunogen H5bis-KLH:H₂N-CYG-VTALWGKVNVDEVGGK-CONH₂ (SEQ ID NO:29) or H1-KLH:H₂N-VHLTPEEKSAVTALW-C—CONH₂ (SEQ ID NO:23).
 9. The immunoassaycomposition of claim 4, further comprising a monoclonal antibody thatselectively binds to hemoglobin variant HbD and glycated HbD and israised against an HbD immunogen H₂N-CYG-VLAHHFGKQFTPPVQAA-CONH₂ (SEQ IDNO:32) or H₂N-QFTPPVQAAYQKVVAGV-GYC—CONH₂ (SEQ ID NO:33); and amonoclonal antibody that selectively binds to hemoglobin variant HbE andglycated HbE and is raised against an HbE immunogenH₂N-CYG-VTALWGKVNVDEVGGK-CONH₂ (SEQ ID NO:29).
 10. The immunoassaycomposition of claim 9, further comprising a monoclonal antibody thatselectively binds to a hemoglobin beta chain, wherein the antibody bindsto a hemoglobin beta chain minimal epitope ¹¹VTALW¹⁵ (SEQ ID NO:17) andis raised against a hemoglobin beta chain immunogen H5bis-KLH:H₂N-CYG-VTALWGKVNVDEVGGK-CONH₂ (SEQ ID NO:29) or H1-KLH:H2N-VHLTPEEKSAVTALW-C—CONH₂ (SEQ ID NO:23).